Title

Author

Date of Award:

Document Type:

Degree Name:

Doctor of Philosophy (PhD)

Department:

Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Sciences

Advisor/Chair:

Jeffery R. Broadbent

Abstract

Amino acids derived from the degradation of casein in cheese serve as precursors for the generation of desirable and undesirable flavor compounds. Microbial degradation of aromatic amino acids is associated with the formation of aroma compounds that impart putrid-fecal, barny-utensil, and floral off-flavors in cheese, but pathways for their production had not been established. This study investigated Tyr and Phe catabolism by Lactobacillus casei and Lactobacillus helveticus cheese flavor adjuncts under simulated Cheddar cheese-ripening conditions (pH 5.2, 4% NaCl, 15°C, no sugar). Enzyme assays of cell-free extracts and micellar electrokinetic capillary chromatography of supernatants indicated that L. casei and L. helveticus strains catabolize Tyr and Phe by successive transamination and dehydrogenation reactions. Major products of Tyr and Phe catabolism included off-flavor compounds formed by chemical degradation of the α-keto acids, produced by transamination, and aromatic α-hydroxy acids derived from α-keto acids by α-hydroxy acid dehydrogenases. Action of Lacrococcus lactis aminotransferase enzymes on Trp, Tyr, and Phe also leads to the formation of α-keto acids, but unlike lactobacilli, the former bacteria do not express dehydrogenase activity under cheese-like conditions (pH 5.2, 4% NaCl, 15°C, no sugar). Since aromatic α-keto acids may degrade spontaneously into undesirable flavor compounds, α-hydroxy acid dehydrogenases may be useful in controlling off-flavor development via diversion of chemically labile α-keto acids to more stable a-hydroxy acids. To test this hypothesis, we investigated the effect of D-hydroxyisocaproate dehydrogenase overexpression by a L. casei adjunct on chemical and sensory properties of reduced-fat Cheddar cheese made with and without addition of 20 mM α-ketoglutarate. The D-hydroxyisocaproic acid dehydrogenase gene (D-HicDH) was cloned into a high copy number vector pTRKH2 and transformed into L. casei ATCC334. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter + L. casei ATCC334 with pTRKH2, and starter + L. casei ATCC334 with pTRKH2: D-HicDH, and then volatile analysis was performed by gas chromatography and mass spectrometry. Statistical analysis of volatile data after 3 mo of ripening at 7°C showed profiles of ketones, aldehydes, alcohols, esters, sulfur compounds, and benzaldehyde were significantly altered by culture treatments and α-ketoglutarate addition, and these treatments also affected sensory flavor attributes of experimental cheeses. Results also indicated overexpression of D-hydroxyisocaproic acid dehydrogenase can divert labile α-keto acids into more stable compounds, but the overall effect seemed to diminish both beneficial and detrimental flavor notes.